Modulating ER-stress and protein aggregation in seeds

Cereal seeds are beneficial for the production of recombinant pharmaceutical proteins (termed molecular farming) because they have evolved to support the accumulation and storage of complex proteins in a stable environment. However, overloading the endomembrane system of the cell, which transports proteins in the seed and distributes them to their final storage compartments, can saturate the protein folding machinery and lead the accumulation of unfolded proteins that aggregate as insoluble inclusions, reducing the yields of functional protein that can be extracted. Plants respond by inducing the unfolded protein response (UPR). This reduces stress on the endomembrane system, but inhibits protein synthesis generally to alleviate the stress at source. The yields of recombinant proteins in seeds could be increased by selectively promoting the folding of extant proteins without inhibiting protein synthesis but this requires a detailed understanding of the UPR and its regulation. We will investigate the molecular basis of the UPR in rice and Arabidopsis to develop strategies allowing the selective activation of ER-assisted protein folding (ERAF). This will be achieved by monitoring ER- stress pathway components in rice and Arabidopsis seeds expressing recombinant proteins and using genomicranscriptomic analysis to elucidate the regulatory network. This information will allow us to test different components for the modulation of the UPR and identify effective strategies to enhance recombinant protein expression. These will also be tested in cereal seeds depleted of endogenous storage proteins, which can interact with recombinant proteins and encourage aggregation. Our results will help to provide generic strategies for the production of high protein yields in the context of molecular farming and will also have wider ramifications in other areas of plant biotechnology, including the development of stress tolerant cereal plants in agricultural settings.

Recombinant proteins produced in the endoplasmic Reticulum (ER) of plant seeds frequently induce intracellular ER-stress and this can result in the formation of insoluble aggregates. This leads to a yield loss preventing the full exploitation of the host cells` transcriptional and translational capacity for these proteins, likely because the capacity of the folding machinery is insufficient and overwhelmed either by the increased amount of endomembrane-passing proteins or by the additional burden of dealing with a protein with a high demand in chaperon-assisted folding. In this research project we investigated Arabidopsis seeds producing different recombinant proteins and we could show that the formation of insoluble ER-derived bodies reveals similarities to the formation of ER-derived protective organelles found in mammalian cells. The newly formed bodies were enclosed by ER membranes and were morphologically similar to Russell bodies found in mammalian cells. 3D electron microscopy revealed that these structures have a spheroidal shape. Similarities in the formation of Russell-like bodies and the plant-specific protein bodies formed by prolamins in cereal seeds could also be established by crossing plants containing ectopic ER-derived prolamin protein bodies with a line accumulating a recombinant protein in Russell-like bodies. Our data clearly demonstrate that the formation of Russell-like bodies in plant seeds occurs with several different recombinant proteins and is therefore a common bottleneck limiting the yield of soluble protein in molecular farming. Our work has led to a better understanding of the cellular and molecular stress response upon recombinant protein production in plants and has formed the basis for developing strategies to counteract ER-stress in plant production hosts. This approach is currently also extended to other plant species such as Nicotiana benthamiana. Our project also produced some significant technical advances: to study the morphology of Russell-like bodies and their influence on endogenous storage organelles we applied 3D electron microscopy techniques including electron tomography and serial blockface electron microscopy to visualize the endomembrane system and ER-derived storage organelles across whole cellular volumes in plant seeds.